CATALYTIC ACTIVITY OF ENZYMES
IN LIVING MATERIALS LAB
INTRODUCTION:
Hydrogen Peroxide (H2O2) is a chemical that is continually formed as a by-product of reactions in living cells. It is poisonous, so the cell must break it down immediately or be destroyed. How does the cell break it down? A catalyst aids this reaction, breaking down hydrogen peroxide.
A catalyst acts to speed up the rate of a chemical reaction. It is not consumed or destroyed during the reaction. Catalysts are present in nearly every chemical reaction in living organisms. Catalysts in living cells are called ENZYMES. Enzymes are protein molecules. In this lab, you will study the breakdown of hydrogen peroxide in a test tube using the enzyme CATALASE.
OBJECTIVE:
We will compare the reaction rate of catalase, found in the tissue of a potato, with a non-protein catalyst called Manganese Dioxide (MnO2). We will examine the activity of catalase under various conditions.
MATERIALS:
MnO2 Powder Three water baths (ice, warm, boiling)
3% Hydrogen Peroxide Solution Fine sand
Pieces of Potato Matches
Liver pieces NaOH solution (0.1 N)
Mortars and Pestles HCl solution (0.1 N)
Test Tubes pH paper
Splints
PROCEDURES:
You will be given a data table on which you will record the reaction rates for each of the six parts of the lab. You will rate them from a 0 to 8, least bubbles to most bubbles.
PART I: HOW DOES A NON-PROTEIN CATALYST AFFECT HYDROGEN PEROXIDE BREAKDOWN?
Ø Put 2 cm of H2O2 in the bottom of TWO test tubes
Ø To one, add a small amount of sand
Ø To the other, add a small amount of MnO2
Ø Immediately hold your thumb over this tube and rate reaction on chart
Ø Insert a glowing splint into the mouth of tube, observe the splint
Ø What happened?
Ø What gas is being produced by the reaction?
2H2O2 2H20 + O2
PART II: HOW DOES AN ENZYME AFFECT HYDROGEN PEROXIDE BREAKDOWN?
A. Zymase in potato
Ø Into one clean test tube, pour 4 cm. of H2O2
Ø Add two pieces of diced potato
Ø Hold your thumb over the tube as you observe the reaction and rate
Ø Insert a glowing splint and observe. What is the gas produced?
Ø Rate the reaction on your chart
Ø How does this reaction compare with the one using MnO2?
B. Zymase in liver
Ø One clean test tube, pour 4 cm of H2O2
Ø Add two pieces of diced liver
Ø Hold your thumb over the tube as you observe the reaction rate
Ø Test with a glowing splint once more
Ø Rate the reaction rate on your chart
Ø Very important.Wait until the reaction is complete (no more bubbles formed)
Ø Very important!! Save contents for the next part of the experiment
PART III: CAN YOU RE-USE AN ENZYME?
Ø Into two clean test tubes, split the used H2O2 from part II by pouring off
Ø Remove the two used pieces of liver and put one in each new tube
Ø Into tube #1, add a new piece of liver and rate reaction
Ø Into tube #2, pour 2 cm. of fresh H2O2 in with the used liver
Ø Observe and rate reaction on chart
PART IV: HOW DOES PARTICLE SIZE AFFECT REACTION RATE?
Ø Into a mortar, add 5-6 pieces of cubed liver and some sand
Ø Grind until it is a paste
Ø Into one test tube, place about ¼ tsp. of crushed liver
Ø Save the remaining potato for the next two parts
Ø Add 2 cm of H2O2
Ø Observe and rate the reaction
PART V: HOW DOES TEMPERATURE AFFECT ENZYME’S ACTION?
Ø Place ¼ tsp of crushed liver in a clean test tube
Ø Place tube in boiling water bath for 5 minutes
Ø Add 2 cm of H2O2 and rate the reaction
Ø Into two clean test tubes, place 2 cm of H2O2
Ø Place one in an ice bath for 5 minutes
Ø Place the other in a warm water bath for 5 minutes
Ø Remove them and add ¼ tsp. of crushed liver to each
Ø Observe and record the rates of reaction
PART VI: HOW DOES pH AFFECT THE REACTION RATE?
Ø Into three clean test tubes place ¼ tsp. of crushed liver
Ø Add 2cm of tap water to one tube
Ø Add 2cm of NaOH solution to tube #2
Ø Add 2 cm of HCl solution to tube #3
Ø To all three tubes, add 2cm of H2O2
Ø Observe and rate reactions on chart
Ø Instructor will check the pH of one group’s three tubes and provide the data for the whole group. Record this information on chart
CONCLUSION AND DISCUSSION:
1) Using your data table, fill in the columns to make a bar graph of your reaction chart. Neatness counts.
2) Examine your results. Can catalysts other than those found in living tissue break down hydrogen peroxide? Explain.
3) When hydrogen peroxide is exposed to the enzyme in the liver, what is changed in the reaction, the enzyme or the hydrogen peroxide, or both?
4) Examine your data. How does the size of the liver particles affect the reaction rate?
5) Similarly, describe how temperature affects the reaction rate.
6) Describe the effect of pH on the reaction rate.
7) If the liver-sand mixture is removed from the test tube, what substances do you think remain in the liquid portion?
8) Is the poisonous hydrogen peroxide produced in living cells transformed into harmless materials by the action of the enzyme catalase? Explain.
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